Effects of rare earth ions on the conformational stability of anticoagulation factor II from Agkistrodon acutus venom probed by fluorescent spectroscopy

Abstract

Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein in a Ca(2+)-dependent fashion with marked anticoagulant activity. The equilibrium unfolding of rare earth ions (RE(3+))-reconstituted ACF II in guanidine hydrochloride (GdnHCl) solution was studied by fluorescence. The GdnHCl-induced unfolding of RE(3+) (Nd(3+), Sm(3+), Eu(3+), Gd(3+))-reconstituted ACF II follows a three-state transition with a stable intermediate state. Substitutions of the RE(3+) ions for Ca(2+) in ACF II decrease the conformational stability of its native state but markedly increase the conformational stability of its intermediate state. The free energy change of RE(3+)-ACF II from the intermediate state to denatured state linearly increases with the increase of ionic potentials of bound metal ions (Ca(2+), Nd(3+), Sm(3+), Eu(3+), and Gd(3+)). The refolding of ACF II from the unfolded state to the intermediate state can be induced merely by adding 10 microM RE(3+) ions without changing the concentration of the denaturant. The kinetic results of the RE(3+)-induced refolding provide evidence indicating that the intermediate state of RE(3+)-ACF II consists of at least two refolding phases and that the refolding rate constant values of the faster phase decrease with the increase of the difference between the radii of Ca(2+) and RE(3+), but the refolding rate constant values of the slower phase are similar to each other. The results of this study indicate that the size of metal ion is the major factor responsible for the metal ion-induced conformational stabilization of the native ACF II, while the metal ionic potential plays a predominant role in stabilizing the conformation for the intermediate state.

The Evolution of New Catalytic Mechanisms for Xenobiotic Hydrolysis in Bacterial Metalloenzymes

“…[118][119][120][121] It has been shown that the apo-form of a metalloenzyme is more unstable than the bound form, [122,123] where the addition of native metal ions increases stability. [75,124] For example, the addition of cobalt or zinc to carbonic anhydrase refolds the molten globule state; it has been proposed that metal ions may associate with a folding intermediate to prevent aggregation pathways for this enzyme. [119,125] Furthermore, different metal ions have been shown to contribute differently to the stability of bovine carbonic anhydrase, highlighting the specificity with which metal ions contribute to stability and folding pathways.…”